Titin and obscurin: giants holding hands and discovery of a new Ig domain subset.

The sarcomere, the fundamental unit of muscle contraction, contains some extraordinarily large polypeptides (0.6–3 MDa). Most of these proteins belong to the titin family, which was initially defined by the sequences of Caenorhabditis elegans twitchin [1,2] and human titin [3,4]. These proteins consist of multiple immunoglobulin (Ig) and fibronectin type III domains (Fn3), one or two protein kinase domains and, in some cases, highly elastic unique regions. As exemplified by studies of titin, the Ig domains serve as binding sites for other proteins and help assemble the sarcomere (reviewed in Ref. [5]). The newest member of the titin family is obscurin (Obs) (~800 kDa), which was initially identified as a ligand of a Z-disk portion of titin [6,7], although it primarily localizes to the sarcomeric M-band in mature muscle [6,8]. Using the power of C. elegans molecular genetics, we described the essential roles of the Obs homolog, UNC-89, in signaling and scaffolding in the M-band 5 years before Obs was discovered [9]. UNC-89 is required for the assembly or stability of the M-band and for thick filament organization [10–13]. The M-band is the portion of the sarcomere where myosin-containing thick filaments are cross-linked by a protein network that, in vertebrate muscle, consists of, at least, the C-terminal portion of titin, myomesin (also a poly-Ig/Fn3 protein) and Obs. The linkage between myosin thick filaments occurs through antiparallel dimers of myomesin that interact via their C-terminal Ig domains and bind to myosin via their N-terminal Ig/Fn3 domains [14,15]. Obs binds myomesin and titin but it is also believed to act as a linker between myofibrils and the SR (sarcoplasmic reticulum). This was first suggested by finding that the C-terminus of some isoforms of Obs interacts with the SR membrane proteins, small ankyrin-1 isoform 5 (sAnk1.5) and ankyrin-2 (Ank2) [16,17]. The Obs knockout mouse supports this model: it shows changes in longitudinal SR architecture even though it has normal sarcomeric organization somewhat surprisingly [18]. The role of Obs in linking the sarcomere to the SR is likely conserved in nematode UNC-89. Although ankyrin-like molecules have not yet been reported to interact with UNC-89, there is genetic evidence for this function: UNC-89 is required for the proper organization of the ryandonine receptor and SERCA, as well as for optimal calcium signaling [19]. Moreover, independent studies on nematode UNC-89 [13] and mouse Obs [20] demonstrate an evolutionarily conserved and novel mechanism by which UNC-89/Obs regulates ubiquitin-mediated protein degradation at the M-band, and this involves inhibition of a cullin 3 complex. The ultrastructural and molecular architecture of the M-line is not as well described for C. elegans muscle as it is for vertebrate muscle. Although C. elegans has an obscurin homolog, UNC-89, there are no obvious homologs of myomesin or titin. TTN-1 is a 2.2-MDa polypeptide localized to the I-band with extension into the outer edge of the A-band and can be regarded as a hybrid between invertebrate twitchin and vertebrate titin [21,22]. This is likely due to the much larger sarcomeres of nematode and many other invertebrate muscles. Moreover, some M-line proteins appear to be nematode specific (e.g., UNC-98 [23] and UNC-96 [24]). An interacting network of proteins (including UNC-89) has been described for the M-line, many having vertebrate homologs/orthologs, which link the muscle cell membrane beginning with integrin through proteins that interact directly with myosin heavy chains in the thick filaments [25–27]. Vertebrates, but not invertebrates, contain genes that encode two additional UNC-89/Obs type proteins: SPEG (striated muscle preferentially expressed gene [28,29]) and Obsl1 (obscurin-like 1 [30,31]). SPEG